SUMO-2 Orchestrates Chromatin Modifiers in Response to DNA Damage Ivo A. Hendriks, Louise W. Treffers, Matty Verlaan-de Vries, Jesper V. Olsen, Alfred C.O. Vertegaal  Cell Reports  Volume 10, Issue 10, Pages 1778-1791 (March 2015) DOI: 10.1016/j.celrep.2015.02.033 Copyright © 2015 The Authors Terms and Conditions

Cell Reports 2015 10, 1778-1791DOI: (10.1016/j.celrep.2015.02.033) Copyright © 2015 The Authors Terms and Conditions

Figure 1 Generation of Cell Lines Stably Expressing FLAG-Tagged SUMO-2 and Purification of FLAG-SUMO-2 Conjugates following MMS Treatment (A) HeLa cells were infected with a lentivirus encoding FLAG-SUMO-2. Cells stably expressing low levels of FLAG-SUMO-2 were selected by flow cytometry. Total lysates were analyzed by immunoblotting to confirm the expression of FLAG-SUMO-2. Ponceau-S staining is shown as a loading control. (B) Stable cell lines were investigated by z-stacked confocal fluorescent microscopy to confirm nuclear localization of FLAG-SUMO-2. Characteristic SUMO nuclear bodies are indicated with arrows. Upon MMS treatment (0.02% for 90 min), SUMO nuclear bodies dispersed. Scale bars represent 5 μm. (C) Schematic representation of the SILAC proteomics workflow. One set of parental HeLa cells and two sets of HeLa cells expressing FLAG-SUMO-2 were differentially SILAC labeled (K0R0/K4R6/K8R10). One labeled FLAG-SUMO-2 set was treated with 0.02% MMS for 90 min. The experiment was repeated with reversal of SILAC labels. (D) Coomassie analysis of total lysate sample versus FLAG-purified SUMOylated proteins, prior to in-gel digestion and analysis by mass spectrometry. (E) Schematic representation of the label-free proteomics workflow for identification of SUMO sites. Four sets of His10-SUMO-2-K0-Q87R cells were treated with 0.02% MMS for 90 min, and four sets were control treated, prior to SUMO site enrichment, identification, and quantification. Cell Reports 2015 10, 1778-1791DOI: (10.1016/j.celrep.2015.02.033) Copyright © 2015 The Authors Terms and Conditions

Figure 2 Analysis of the Mass Spectrometry Data Reveals Highly Enriched Interaction Clusters (A) Overview of proteins identified by the SILAC protein-level workflow, by the label-free sites-level workflow, and proteins in which sites were identified. Only proteins that were found by both approaches, shaded in blue, were used for further analysis. (B) Scatterplot analysis of protein SUMOylation ratio (log2) in response to MMS, versus protein SUMOylation SILAC ratio (log2) enrichment over the parental control. The parental control refers to cells not expressing FLAG-SUMO-2. “Dynamic” proteins were found consistently and significantly changed across all proteomic analyses and replicates. Proteins of interest that were confirmed by immunoblotting analysis are indicated. (C) Volcano plot displaying SUMOylation site ratio (log2) in response to MMS versus the negative p value (log10) resulting from permutation-based false discovery rate. Red dots indicate downregulated sites; blue dots indicate upregulated sites. Sites in proteins of interest are indicated. (D) Term enrichment analysis of identified up- and downregulated SUMOylated proteins in response to MMS. Relative score of regulated SUMOylated proteins versus the human proteome is indicated for the Gene Ontology (GO) Molecular Functions, Cellular Compartments, and Biological Processes categories. A full term enrichment analysis is available for upregulated SUMO targets (Table S8) and downregulated SUMO targets (Table S9). (E) STRING network analysis of all regulated SUMOylated proteins in response to MMS. Enrichment ratio refers to the amount of interactions observed versus the amount of expected interactions (p > 0.4). Cell Reports 2015 10, 1778-1791DOI: (10.1016/j.celrep.2015.02.033) Copyright © 2015 The Authors Terms and Conditions

Figure 3 Confirmation of Mass Spectrometry Results (A) HeLa cells and HeLa cells stably expressing FLAG-SUMO-2 were either mock treated or treated with MMS. Cells were lysed, and FLAG-IP was performed to enrich SUMOylated proteins. Total lysates and SUMO-enriched fractions were analyzed by immunoblotting to confirm the SUMO regulation of the indicated proteins. (B) Immunoblotting verification of overall level of SUMO-conjugated proteins in the total lysates and SUMO-enriched fractions after MMS treatment and FLAG-IP. (C) Cell lines stably expressing FLAG-SUMO-2 were investigated by confocal fluorescent microscopy for the presence of PML nuclear bodies. Upon MMS treatment, PML bodies rapidly dispersed. Scale bars represent 5 μm. Cell Reports 2015 10, 1778-1791DOI: (10.1016/j.celrep.2015.02.033) Copyright © 2015 The Authors Terms and Conditions

Figure 4 SUMOylated JARID1B and PML Are Rapidly Degraded by the Proteasome upon MMS Treatment U2OS cells stably expressing His10-tagged SUMO-2 were treated with 0.02% MMS for the indicated amount of time. One set of cells was additionally treated with the proteasome inhibitor MG132 at 10 μM. Cells were lysed, and His10 pull-down was performed to enrich SUMOylated proteins. Total lysates and SUMO-enriched fractions were analyzed by immunoblotting using antibodies against JARID1B and PML. Ponceau-S staining was performed on total lysate fractions as a loading control. Cell Reports 2015 10, 1778-1791DOI: (10.1016/j.celrep.2015.02.033) Copyright © 2015 The Authors Terms and Conditions

Figure 5 Degradation of SUMOylated JARID1B and PML Is Mediated by the SUMO-Targeted Ubiquitin Ligase RNF4 (A) U2OS cells stably expressing His10-tagged SUMO-2 were treated with 0.02% MMS or 1 μM arsenic trioxode for 90 min or 4 hr, respectively. One set of cells was additionally depleted for RNF4 by shRNA-mediated knockdown. After treatment, cells were lysed, and SUMOylated proteins were enriched. SUMO-enriched fractions were analyzed by immunoblotting using the indicated antibodies. Total lysate fractions were analyzed by immunoblotting for depletion of RNF4. (B) U2OS cells were treated with MMS for the indicated amount of time. One set of cells was additionally depleted for RNF4 by shRNA-mediated knockdown. Groups of cells were investigated by z-stacked confocal fluorescence microscopy for the presence of PML bodies. Scale bars represent 50 μm. (C) Multiple fields of cells were recorded as in Figure 5B. Cells were counted, and PML bodies were quantified in every cell and scored for the presence of at least five PML bodies. The number of quantified cells is indicated below each experiment. Error bars represent 2 × SEM. ∗∗p < 1 × 10−15 Cell Reports 2015 10, 1778-1791DOI: (10.1016/j.celrep.2015.02.033) Copyright © 2015 The Authors Terms and Conditions

Figure 6 H3K4me2/3-Specific Demethylase JARID1C Is Recruited to the Chromatin in Response to DNA Damage (A) U2OS cells were treated with an increasing dose of MMS for 18 hr. Cells were lysed and analyzed by immunoblotting with the indicated antibodies. (B) U2OS and HeLa cells were treated with 0.02% MMS for the indicated amount of time, and one set of cells was additionally depleted for RNF4 using siRNA-mediated knockdown. Cells were lysed and analyzed by immunoblotting with the indicated antibodies. (C) U2OS cells were mock treated or treated with 0.02% MMS for 90 min, and subsequently cellular fractionation was performed. Cellular fractions and total lysates were analyzed by immunoblotting for JARID1B and JARID1C. Ponceau-S staining of the histones is shown as a loading and fractionation control. (D) As in (C), with one set of cells additionally depleted for RNF4 using shRNA-mediated knockdown. Additionally, total lysates were analyzed by immunoblotting for H3K4me3 and RNF4. Cell Reports 2015 10, 1778-1791DOI: (10.1016/j.celrep.2015.02.033) Copyright © 2015 The Authors Terms and Conditions

Figure 7 JARID1C Demethylates Global H3K4me3, and SUMOylation Mediates Chromatin Remodeling and Transcriptional Regulation, in the Cellular Response to Alkylating DNA Damage (A) U2OS cells were transfected with GFP-JARID1C and either mock treated or treated with 0.02% MMS for 90 min. Subsequently, cells were fixed and investigated by confocal fluorescent microscopy to check for the presence of JARID1C (GFP) and the level of H3K4me3. Scale bars represent 25 μm. (B) Schematic overview of the effect of SUMOylation on various key proteins involved in the cellular response to DNA damage and the downstream effect on chromatin remodeling and transcriptional regulation. Cell Reports 2015 10, 1778-1791DOI: (10.1016/j.celrep.2015.02.033) Copyright © 2015 The Authors Terms and Conditions